Viruses, in particular adenoviruses, are potentially useful vectors for vaccination and/or gene therapy. Recombinant adenoviruses are a preferred class of viral vectors for use in vaccination or gene therapy. Robust and high yield processes for large scale manufacturing are needed to support the world with sufficient amounts of (recombinant) vaccines to combat infectious diseases. In support of commercial scale production reliable analytical methods are required to monitor the quantities of (adeno)viruses throughout the manufacturing process (from upstream processing to downstream processing) to ensure the safety, efficacy and quality of (adeno)virus-based vaccines/therapies.
In addition, during process development and for production of adenovirus-based clinical trial material, samples are analysed in order to either monitor or control the production process and the safety of the product. One of the analyses typically performed on such samples is the determination of the total concentration of intact adenovirus particles. Current methods with limited suitability for this purpose are quantitative polymerase chain reaction (qPCR) and anion exchange chromatography (AEX-HPLC). qPCR is based on DNA amplification of a region specific for the adenovirus and fluorescence detection of the formed amplicon. Quantitative PCR is an expensive (cost price of chemicals) and laborious technique. The total time from sampling to result reporting can be up to three days, since numerous replicates are required for the results to have acceptable precision. An additional drawback is that qPCR determines the concentration of adenovirus particles indirectly by determining the DNA content of the particles instead of determining the intact particles themselves.
AEX-HPLC with UV detection makes use of a charge-based separation of intact adenoviruses from sample matrix components. Quantification is performed by external calibration using an adenovirus reference standard. The AEX-HPLC method suffers from adsorption, carry-over and recovery issues for samples containing cell lysate or high salt concentrations, and is therefore not suitable to determine the concentration of adenoviruses accurately and precisely throughout the entire production process.
There thus is a need for an improved method for the quantification of (adeno)viruses. In particular, in order to support process development for (adeno)virus based vaccines, it would be useful to have one single method that allows for direct quantification of (adeno)virus particles throughout the process with high accuracy and precision and with a short time to result, without the drawbacks of for example adsorption, carry-over and/or poor recovery.